A key event in the progression of osteoarthritis (OA) is the loss of proteoglycan aggregate from the extracellular matrix of articular cartilage. In addition to aggrecan, a significant decrease in hyaluronan (HA), the core filament of the cartilage proteoglycan aggregate, is also observed in cartilage derived from OA patients as compared to normal human cartilage. These losses are due to changes in the metabolic state of resident chondrocytes. In past studies we have tested the hypothesis that HA turnover occurs primarily through an endocytosis event, mediated by the HA receptor CD44. Experimental conditions that upregulate the loss of aggrecan and HA, also affect an enhancement in CD44 expression and a coordinate increase in HA endocytosis. In addition we have tested whether chondrocytes also use CD44 to sense and respond to a loss of proteoglycan by initiating signaling cascades. Until recently we have only been able to examine CD44 functions by using chondrocyte cell-culture studies. The central hypothesis of this new proposal is that HA within the extracellular matrix (ECM) of damaged cartilage is limiting. As such, matrix repair and aggrecan retention are ineffective. We have been recently successful at introducing human transgenes into intact human, bovine and mouse cartilage explants through the use of human adenoviral and adeno-associated virus constructs. This proposal includes several innovative approaches. We will use a gene transfer approach to introduce a HA synthase (HAS2) transgene into intact human OA cartilage explants. This approach will generate a selective and substantial increase in HA within the ECM to address the question of whether an increased deposition of locally-synthesized HA in situ provides for cartilage repair and a diminution of aggrecanolysis biomarkers as resident chondrocytes are coaxed into a state of quiescence. Our second goal will be to determine the mechanism for the HA-induced return to homeostasis and whether HA receptors such as CD44 are responsible. Our long-term goal is to determine the importance of HA to cartilage homeostasis and, how to alter HA metabolism in vivo especially within human articular cartilage.